Gene Expression Signature And Expression Profile Of TLR7 In The Liver Of Biliary Atrsia Mice

Objective:The aim of this research is to identify genes potentially involved in the pathogenesis of bile duct obstruction in biliary atresia(BA).Methods:1 The rhesus rotavirus Balb/c mouse biliary atresia model and Sample collection.The healthy neonatal mice were selected.The pups received an intraperitoneal injection of 1.5×10~6 fluorescence-forming units of RRV in 20μl within 24 h postpartum were considered as BA groups(n=68);and the pups received20μl supernatant of MA104 cell culture medium were considered as NC groups(n=72).Record the weight and survival of the mice daily.Liver,extrahepatic bile duct and serum samples were harvested from BA and normal control(NC)groups at 1,3,5,7,10 and 14 days postinoculation.2 The levels of serum total bilirubin(STB)and conjugated bilirubin(CB).The levels of STB and CB in serum in the two groups at each time points were measured by using kits according to the manufacturer’s instructions.3 Hematoxylin and eosin(HE)staining.HE staining of liver tissues and extrahepatic bile duct specimens were conducted.4 Chemokines in serum and liver.The expression of 12 chemokines in serum and liver at different time points were detected by flow cytometry by using kits.5 Differential screening and bioinformatics analysis of liver expression gene and microRNA.Liver specimens were obtained from the 7th day after the injection.Total RNA was isolated from the liver tissues of BA and the normal liver tissues.RNA was purified with QIAGEN RNeasy mini Kit,converted to c DNA and hybridized using Affy GeneChip Mouse Gene 1.0 ST Array and Affy GeneChip miRNA 4.0 Array.Meanwhile,bioinformatics analysis including differentially expressed genes screening,series test cluster of gene ontology,and gene signal network.6 The role of TLR7 signaling pathway in the pathogenesis of biliary atresia mice.Expression of TLR7 on biliary epithelium was measured by immunohistochemical staining,western blotting and real-time polymerase chain reaction.Activation of phosphorylated interferon(IFN)regulatory factor(IRF)7 was shown by immunoblotting and expression of IFN-β,interleukin(IL)-1α,and IL-6were measured using flow cytometry.7 Statistical analyses.All analyses were performed in a double-blinded manner.Student’s t-test and one-way analysis of variance(ANOVA)followed by Bonferroni’s test were used for single and multiple comparisons,respectively.Pearon correlations were calculated using SPSS19.0.Values of p﹤0.05 were considered ststistically significant.Results:1 The rhesus rotavirus Balb/c mouse biliary atresia model and Clinical symptoms.Pups injected with RRV developed the clinical signs of biliary obstruction:jaundice,bilirubinuria,and alcoholic stools.2 The levels of total bilirubin(TB)and conjugated bilirubin(CB).Detection of serum bilirubin showed that serum total bilirubin began to increase and gradually increased on the day 5,and serum conjugated bilirubin began to increase and gradually increase on the day 7.3 The results of HE staining.The results of HE staining showed that inflammatory infiltration occurred in liver in the 5th day,and the results in intrahepatic bile duct disappeared in the day 10 and 14.The results of bilirubin test showed that serum total bilirubin and direct bilirubin began and gradually increased in the day 5 and the day 7,respectively.4 The results of chemokines.The results of chemokines test showed that:compared with the control group,in addition to the expression level of CCL2,CCL20and CXCL9 in serum of the mice with BA,that of the other chemokines in liver were significantly up-regulated.Among them,CCL2,CCL4,CXCL1 and CXCL11 started to be up-regulate from the 3rd day,which was earlier than serum total bilirubin.CCL3,CCL4,CCL5,CCL20,CXCL1,CXCL10 and CXCL19 started to be up-regulated from the 7th day.5 The microarray analyses.There are 254 different genes which were associated with biological process between BA and NC group.By the data analysis derived from microarray,the function of the differentially expressed genes mainly involved in innate immune response,response to virus,defense response to virus,oxidation-reduction process,metabolic process,cellular response to interferon-beta,negative regulation of viral genome replication immune response and so on.Further analysis of gene pathway network revealed that the expression of genes in the TLR signaling pathway and apoptosis were totally unregulated.Therefore,the22 genes of innate immune response were ranked by p value;the TLR7 was the most significant and its downstream gene IRF7 also had a certain degree of up-regulation.6The role of TLR7 signaling pathway in the pathogenesis of biliary atresia mice.Immunohistochemical staining revealed that there were no differences between the NC and BA groups on day 3;the biliary epithelium was TLR7 immunopositive in the BA group on days 5 and 7;TLR7 immunopositive inflammatory cells were evidently infiltrated around the sinusoid and bile epithelial integrity was destroyed on days 10and 14.Western blotting and real-time polymerase chain reaction revealed that biliary epithelium was immunopositive for TLR7 and TLR7 immunopositive inflammatory cells were evidently infiltrated around the sinusoid on days 5–14 when the bile ducts became obstructed.The downstream gene of TLR7 signaling,Phosphp-IRF7,was active and cytokines were released at the same time.After TLR7 silencing,the cytopahogenic effect(CPE)of the human intrahepatic bile duct epithelial cells infected with the RRV were earlier and the disease was changed seriously.Conclusion:CCL2,CCL4,CCL11 and CXCL1 were up-regulated earlier and participate in the occurrence of BA.CCL3,CCL5,CCL20,CXCL9 and CXCL10were up-regulated later and involved in the disease development.Therefore,it was speculated that the disease could be evaluated by multi-index serological detection and early evaluation of the disease can be conducted.The pathogenesis of the biliary atresia is related to the immune response,and the innate immunity has a significant effect.By in vivo study,TLR7 signal pathway was up-regulated in BA group;by additional in vitro study,intact TLR7 signal pathway might have some protective abilities in BA pathogenesis.